Viability assessment of Chlamydia trachomatis in men who have sex with men using molecular and culture methods.

Abstract

Objectives: Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection (STI) worldwide. Diagnosis relies on nucleic acid amplification techniques, such as PCR, which does not distinguish between viable pathogens and residual bacterial DNA, leading to potential overdiagnosis and overtreatment. PCR with confirmation of pathogen viability has not been widely explored in the STI field. Our aim was to establish a C. trachomatis viability PCR (V-PCR) and to apply it to anorectal swabs from men who have sex with men (MSM).

Methods: We performed validation of a published V-PCR protocol by preparing artificial samples with known ratios of viable and non-viable C. trachomatis. Mock samples were treated with propidium monoazide (PMAxx^TM) before DNA extraction and quantitative PCR (qPCR) to detect C. trachomatis. The V-PCR was then applied to C. trachomatis PCR-positive anorectal swabs from MSM. Viability was expressed as the difference in C. trachomatis (CT) copies between PMAxx^TM untreated and treated samples (ΔLog10 CT/mL). The anorectal samples were inoculated in cell culture for isolation. Genotyping was performed by examining the ompA gene sequence.

Results: Of 236 anorectal swabs, 69 (29.2%) were C. trachomatis PCR-positive, and we obtained V-PCR data from 54. There were 7/54 (12.9%), samples with <1% viable CT (>2.52 ΔLog10 CT/mL) 4/54 (7.4%) samples with 1%-10% viable CT (1.59-2.52), 16/54 (29.6%) with 10.01-50% viable CT (0.86-1.59) and 27/54 (50.0%) with 50.01%-100% viable CT (<0.35-0.86). C. trachomatis was isolated successfully from 39/69 (56.5%) samples in cell culture. Genotypes based on ompA were obtained for 62/69 (89.9%) samples: G (n=15/62), D/Da (n=15/62), J (n=15/62), E (n=11/62), L1 (n=4/62) and L2 (n=2).

Conclusions: We successfully implemented a viability test based on PCR, which can distinguish, detect and quantify viable C. trachomatis in anorectal swabs from MSM. Rapid, reliable assessment of C. trachomatis viability could help to improve antimicrobial stewardship.